Cold air plasma improving rheumatoid arthritis via mitochondrial apoptosis pathway

Abstract Rheumatoid arthritis (RA) has plagued physicians and patients for years due to the lack of targeted treatment. In this study, inspired by the commonality between rheumatoid arthritis fibroblast‐like synoviocytes (RA‐FLS) and cancer cells, the therapeutic effects of cold air plasma (CAP) on RA are studied systematically and thoroughly. In/ex vivo results show that CAP with the proper dosage significantly relieves symptoms including synovial hyperplasia, inflammatory infiltration, and angiogenesis and eliminates the root cause by triggering the self‐antioxidant capability of the surrounding tissue. The mechanism on the molecular and cellular level is also revealed that the spontaneous reactive oxygen species (ROS) cascade induces the mitochondrial apoptosis pathway on RA‐FLS. This study reveals a new strategy for targeted treatment of RA and the mechanistic study provides the theoretical foundation for future development of plasma medicine.

cal effects such as multiple sclerosis and lymphoma. 7,8 Hence, RA has plagued physicians worldwide due to its lack of efficient and targeted treatment.
As early as 1550 BC, cautery was a mysterious and ancient method considered complementary to the treatment of complex diseases. In the 18th and 19th centuries, cautery was reported in Chinese and Indian medicine to improve joint pain in RA. 9 Because of the inability to apply cautery to the intra articular and the inability to control the dose, cautery did not make substantial progress in the treatment of RA. In modern medicine, low-temperature plasma radiofrequency ablation (LTPRA) has similar characteristics to cautery.
The principle of operation is to ablate the local tissue around the electrode by evaporating it with radiofrequency energy, which seems to be a potential method to act on the joint synovium. 10 However, LTPRA inevitably damages the healthy tissue while removing the diseased tissue, and it is difficult for LTPRA to realize the cooperation of visualization system and water supply system in a narrow joint space, which is still not a method to solve RA joint lesions. 11 Recent studies have provided important clues concerning jointtargeted therapy of RA. RA-FLS are the core effector cells of knee joint synovium. The activated RA-FLS cells are the lesion center of RA synovial proliferation. 12 RA-FLS is extremely similar to tumor cells. 13 On the one hand, the defect of RA-FLS apoptosis originates from aberrant proliferation and transformation in tumor samples, which shows similar invasiveness and adhesivity. On the other hand, the imbalance between antiapoptotic and proapoptotic molecules is an important cause for RA-FLS antiapoptosis. 14,15 The above commonality between RA-FLS and tumor cells inspires the attempt to treat RA referring to the potential anticancer strategy.
Cold air plasma (CAP) has been widely explored in the clinical as physical treatment for tumors. Although the reactions between plasma and cells are still not well understood, ROS representing oxygen-containing free radicals in cells including superoxide radical (O 2 •À), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH•), peroxynitrite (ONOOÀ), nitric oxide (NO), and so on in the plasma are considered to be important factors affecting the functions of living cells. 16 CAP induces cytotoxic reaction in many cell lines by regulating the intracellular ROS levels. 17 In this respect, CAP effectively modifies glycated GPx, increases the enzymatic activity, and reduces oxidative stress in diabetic mice. 18 In addition, CAP is used to induce apoptosis in glioblastoma, cervical cancer, and breast cancer cells. 19 Therefore, CAP is expected to inhibit abnormal cell activation such as RA-FLS.
However, the preclinic feasibility of using a plasma to treat RA and whether it can regulate oxidative stress or promote apoptosis of RA-

| CAP devices and plasma characterization
The CAP devices required for the in vivo and ex vivo experiments were designed and fabricated according to the target application.
CAP used to intervene with the joint cavity of rats (CAP-i) is shown in Figure 1a. The voltage difference between the puncture needle and tissue ionizes the air to generate the plasma, which is brought to the vicinity of the intraarticular synovium. The advantage of this design is to avoid the direct interaction between the electrode and intraarticular synovium. In addition, the cathode is connected to the rats at a zero potential and the device is operated at a peak current of only 4 mA, which is so small that there is very little tissue damage. The ex vivo experiments are conducted in RA-FLS using the plasma device (CAP-e) shown in Figure 1b, which is designed individually to achieve high-throughput treatment of 96-well cell culture plates. The detailed fabrication process of the device can be found elsewhere. 20 Although the devices for the in vivo and ex vivo studies vary in appearance, they share key parameters such as the operating temperature, discharge form, and excited-state generation. The temperature of the plasma generated by these two devices is monitored by an infrared thermometer as shown in Figure 1c. After the CAP-e acts for 30 s, the temperature of the discharge panel is 29.0 C, and increases gently to 30.4 C after 120 s. When the CAP-i is discharged to 120 s, the temperature of the arc column is maintained at 25.3 C and the temperature of the dielectric layer does not exceed 32.6 C. These results indicate that both devices establish a working environment with a temperature less than 40 C thus ensuring the safety of cells and tissues. The moderate working temperature can be explained by the pulse discharge form of the CAP device. The discharge of both CAP-i and CAP-e device is discontinuous and periodically pulsed thereby limiting the temperature rise.
The chemical composition of the plasma is determined by optical emission spectroscopy (OES). 21 This technology captures the unique emission spectrum of discharge plasma by using the characteristic emission spectra of different atoms or molecules. As shown in Figure 1e, the high intensity peaks at 315, 336, and 380 nm in the UV-vis spectrum are related to the second positive system of N 2 and the first negative system of N þ 2 . 22 The characteristic emission peaks of atomic oxygen appear at 777 and 845 nm. 23 These two substances correspond to ionic nitrogen and oxygen respectively, which represent the main part of the air. Moreover, the peak of OH* is observed at 296 nm for both devices. 24 The above results fully illustrate the physical and   The CAP120 group shows only two small color Doppler spots similar to the normal group representing highly improved vascular conditions. To summarize, CAP120 shows preferable therapeutic effects on RA by alleviating the symptoms of synovial hyperplasia, inflammatory infiltration, and angiogenesis.

| Establishment of the animal model
As shown in Figure 2d, the concentration of SOD in the AIA group is at a low level (0.7 U/ml). After the plasma treatment, SOD in the tissue increases to 20 U/ml similar to the normal group indicating

| CAP inhibits the viability and proliferation of single RA-FLS
The morphology of RA-FLS before and after CAP intervention was observed under the microscope. As shown in Figure  These results indicate that both the viability and proliferation of RA-FLS are inhibited by CAP and so development of RA is prevented from the macroscopic perspective.

| CAP obstructs the invasion and migration of RA-FLS to the surrounding tissues and environment
RA-FLS shows "tumor-like" properties with the invasive and migratory behavior, which accelerates exponentially disease deterioration. 27 The migratory behavior is evaluated by the scratch assay. The scratch healing speed of the control (no treated) group is faster as shown by the distance between the edges shortening significantly after 24 h.
The healing speed decreases gradually after the CAP treatment as reflected by the wider distance between the edges in CAP60 and CAP120, which is observed from the figure and quantitatively indicated by halved migration rates in CAP120 compared with the control Relative expression of caspase-3 protein; relative expression of cytochrome c protein. * and ** indicate that there are significant differences between the two groups, which are equal to *p < 0.05 and **p < 0.01 respectively. "ns" indicates that there is no difference between the two groups, which is equal to ns p > 0.05. Before and after CAP intervention, there was no significant difference in the protein content of activated caspase-3 between CAP60 group and control (no treated) group (p = 0.095), and there was no significant difference in the protein content of cytochrome c between CAP60 group and control (no treated) group (p = 0.131).
13.69% and 51.49% of the cells in CAP60 and CAP120, respectively, and the progression is consistent with the morphological results observed by TEM.

| Apoptosis is initiated by excessive and instant oxidative stress
The CAP treatment increases the ROS content in RA-FLS as demonstrated by flow cytometry (Figure 4c).  (Figure 4c). Since ROS in a low concentration impose antiapoptosis effects but in a high concentration induces apoptosis, 31 it is proposed that CAP triggers the programed suicide of RA-FLS by instantaneously exerting excessive oxidative stress.

| Apoptosis is associated with the mitochondrial pathway
Mitochondria is believed to play a central role in the oxidative stressinitiated apoptosis. 32 In the next step, we test the MMP (ΔΨm) to explore the mechanism (Figure 4d). The percentages of C2 and C4 represent the cells under better conditions and a reduced membrane potential, respectively. 33 The scatter plot shows that by prolonging the CAP treatment, the cell community tend to move from C2 to C4 and the ratio of cells with normal MMP decreases from 88% for the control (no treated) to 81% and 70% for CAP60 and CAP120, respectively ( Figure 4d). The results reveal that the CAP treatment causes permeabilization of the mitochondrial membrane verifying the mitochondrial pathway associated apopotosis.
To understand the cascade of events in the mitochondriaassociated apoptosis, the proapoptotic and antiapoptotic proteins are semi-quantified. As shown in Figure 4e, the western blot analysis shows that Bcl-2 playing the antiapoptotic role is downregulated, while Bax as the proapoptotic member is upregulated. The Bcl-2/Bax ratio drops by six times from 1.5 for the control (no treated) to $0.25 for CAP120. As a result, Bcl-2 cannot restrain Bax thus triggering apoptosis 34

| CAP-i and CAP-e equipment configuration
The CAP-i device consists of a gas delivery device, a handle, and two electrode assemblies. The gas delivery device consists of a gas cylinder, a gas valve, and a gas flow meter that controls the amount and rate of gas input to the AIA rat joints. The two electrodes are assembled with an energized needle electrode and a grounded clip electrode is connected to a high-voltage transformer.
The needle electrodes are covered with an insulating film to pre- The detailed preparation process is described in our previous paper. 20 The advantage of this design is to reduce leakage and energy consumption. The working voltage of CAP-e is about 2 kV and the current is about 0.1 mA.

| Intervention of CAP-i on synovium of adjuvant-induced arthritis rats
As early as 2005, Alexandre et al. reported that the treatment of intervertebral disc herniation by injecting oxygen-ozone mixed gas into the intervertebral disc had a significant therapeutic effect on pain and nerve root dysfunction. 55 Studies showed that intraarticular ozone therapy could effectively alleviate the pain of patients with osteoarthritis. 56 Based on these studies, intraarticular gas injection was feasible and safe to some extent. The uppermost end of the puncture needle was connected to the electrode and the puncture needle was covered by insulating materials with the tip exposed only to the discharge as the gas was injected into the body. The other wire was grounded and fixed to the rats' legs. During the experiment, the rat was anesthetized and fixed to the plate with a rubber band.
After the puncture needle was inserted into the rats' joints, the power and timer were on and by using the gas cylinder and flow valve, 2 ml of air was injected slowly into the joint cavity of the AIA rat. Finally, the plasma was generated around the joint synovium by arc discharge.

| Establishment of the AIA rats' model and experimental design
Male SD rats' weighing 240 ± 20 g (7-8 weeks old) were obtained from the Experimental Animal Center of Anhui Medical University and all animal care and treatment procedures were approved by the Animal Ethics Committee of Anhui Medical University. The experimental rats were divided randomly into four groups (eight rats in each group): normal group, AIA group (untreated), CAP 60 (The CAP treatment durations were 60 s), and CAP 120 (The CAP treatment durations were 120 s). After adaptive feeding at room temperature for 1 week without abnormality, all the rats were anesthetized with chloral hydrate (3:1 mg/kg, intraperitoneal). The AIA rats' model was established by subcutaneous injection of the complete Freund' s adjuvant (Chondrex, USA) at the left rear toe (injection dose: 0.1 ml/100 g).
The rats in the normal group were injected with the same amount of normal saline. After modeling, the rats were fed with a normal diet and underwent normal activities for 21 days. On the 21st day, the treatment group was treated with CAP at the knee joint for 60 s or 120 s, once every other day within 7 days for a total of four times.
The rats in normal group and AIA group were not treated. The CAP treatment was completed on the 27th day and 24 h after the last CAP treatment, all rats were sacrificed. The whole experiment lasted for 28 days.

| Doppler ultrasound
On the 28th day, the knee joints of rats in the normal group, AIA group, CAP60 group, and CAP120 group were examined by the

| Wound healing assay
The cells of each group were cultured and the medium was changed to a serum-free one before the experiment followed by addition of

| Statistical analysis
The data generated in the experiments were presented as mean ± standard deviation (SD) and all the data were analyzed statistically by the IBM SPSS software version 23.0 (SPSS). Multigroup data were compared by one-way ANOVA and multiple comparison among groups (ANOVA) was performed by the LSD test. The independent sample t-test was employed to compare the data between the two groups. The p value refers to the statistical probability with p < 0.05 indicating statistically significant difference. funding acquisition (equal); writingreview and editing (equal).